Assays Flashcards
EdU
Proliferation - DNA synthesis
Immunoassay to quantify cells that
have moved through S-phase
Thymidine Analog is incorporated into DNA, Alexa Fluor (488) dye shows fluorescence
1. Add EdU to media & culture cells for determined
amount of time
2. Fix & permeabilize cells
§ Fix: stabilize & preserve cells
§ Permeabilizing: Poking holes in the cells to large molecules
can enter
3. “Click” on fluorescently labeled azide to label the
EdU incorporated into the DNA
4. Use fluorescence microscopy to visualize
fluorescence
5. Analyze images to determine % of actively
proliferating cells
MTS
Biochemical assay to indirectly
quantify viable (respiring) cells
Blue dye undergos reduction and becomes pink with high fluorescence
- Culture cells
- At determined time, add the assay
reagent to the cell culture medium &
incubate (between 0.5-4 hrs) - Measure the amount of resorufin dye
produced by measuring the absorbance
at 570 nm - Process & analyze the data . pink = more living, higher ABS,
Live/Dead
Biochemical assay to
indirectly quantify viable cells
§ Uses two dyes:
* Dye that labels only live cells (ex. Calcein-AM)
* Dye that labels only dead cells (ex. ethidium homodimer-2)
1. Culture cells (treatments & controls).
2. At determined timepoints, add the assay reagents to
the cell culture medium & incubate for a defined period
of time.
3. Use fluorescence microscopy to visualize fluorescence
from live & dead cells. Take images.
4. Analyze images to determine % of viable cells
BCA
Colorimetric assay to determine total protein
concentration in a sample solution
§ Pros: easy, cheap
§ Cons: information about total protein levels
Prepare samples
& standards Prepare working
reagent
Add standards &
samples into wells
Add working reagent
into wells
Seal plate & incubate at
37ºC for 30 mins
Measure absorbance at
562 nm in a plate reader
ELISA
Enzyme-Linked Immunosorbent Assay (ELISA)
§ An assay technique designed for detecting and
quantifying substances such as peptides,
proteins, antibodies, and hormones secreted by
the cell.
Pros:
* Sensitive
* Proteins detected
using native
structure
* Can detect proteinprotein interactions
Cons:
* Detect only 1
analyte at a time
* Cross-reactivity
* False positives
* Time consuming to
make own
Well coated
with capture
antibody
Antigen added Detection
antibody added
Substrate added
& plate read
Enzymeconjugated
detection
reagen
Western Blot
Analytical technique used to detect specific
proteins in a sample of cell or tissue
homogenate
* Determines the presence and integrity of a protein
- Pros:
- Sensitive
- Specific
- Cons:
- Technically
demanding - Time consuming
- Proteins detected in
non-native structure
Order:
Sample Preparation: lyse, quantify, denature, load buffer
Gel
Preparation
Sample
Loading Electrophoresis Protein
Transfer Blocking Primary
Antibody
Secondary
Antibody Visualization
Flow Cytometry
Analyzes the physical and chemical
characteristics of particles in a fluid as they
pass through at least one laser
§ Cell components are fluorescently labelled and
then excited by the laser to emit light at varying
wavelengths
- Pros:
- High throughput
- Fast
- Enumerate cell
subtypes - Look at numerous
parameters
simultaneously - Cons:
- No localization of
antigen - Needs single cells
- Instrument
calibration required
at each use
Immunofluorescence
Uses fixed cells or tissues, not live, determine spatially
where a protein is and relative concentrations.
- Pros:
- Enumerate cell subtypes
- Provides localization of
antigens - Cons:
- Time consuming
- Labor intensive
Use imaging software to
compare:
* Fluorescence
intensity
* More/less
fluorescence (i.e.,
protein) in a sample
vs. another
* Relative locations
of protein
* More/less
fluorescence (i.e.,
protein) in
cytoplasm vs.
nucleus
* More organization
of protein structures
Under-Agarose Assay
Cell migration assay
Suspension placed in a
while of semi solid agarose
* Motile cells crawl on solid
substrate underneath
agarose
